Pancreatic cancer (PC) still remains one of the deadliest of all cancers with almost uniform lethality despite aggressive treatment modalities for the past 60 years. Developing novel strategies to prevent/delay/inhibit progression of PC is currently of intense interest. The latency period from undetectable lesions through progression of precancerous lesions, until clinical diagnosis of multifocal and multiclonal PC can span 15-20 years. This long latency could allow time for intervention once quantitative assessment of PC mechanisms is made and effective chemopreventive agents are developed. Previous studies have shown that insulin signaling drives cancer stem cells (CSCs) toward the aggressive behavior of PC. Recent meta-analysis showed that anti-diabetic drug metformin reduces risk of PC. Metformin affects CSCs by altering signaling through the target of rapamycin (mTOR). Other studies suggest that glutamine is required for the uptake of essential amino acids and in maintaining activation of mTOR kinase. To better design new treatment strategies targeting CSCs against early stage pancreatic cancer, the knowledge of the CSC initiation during precancerous lesion formation is extremely important. In recently published and preliminary studies, we have found that overexpression of glutaminase, an early mediator that stimulates insulin signaling, correlates with PC and that treatment with metformin during lesion progression significantly decreases glutaminase, CSCs and PC. However, whether metformin treatment earlier in the disease initiation might further delay PanIN and CSC initiation is unknown. Given these findings, we hypothesize that glutaminase is overexpressed at the stage of precancerous lesion formation, initiating the CSC proliferation that furthers development of PC. We also hypothesize that metformin will inhibit specifically the initiation of CSCs and formation of pancreatic precursor lesions by regulating glutaminase and insulin signaling molecules, thus inhibiting/preventing PC development. These hypotheses will be addressed in the experiments of the following Specific Aims: 1) to determine whether glutaminase regulates initiation of CSCs and PanIN lesion formation in Kras mutant mice. 2) To determine the effect of metformin on glutaminase regulation affecting CSC initiation and PanIN lesion formation. Should glutaminase regulate insulin signaling and thereby initiation of CSCs at precursor lesion formation, and should metformin treatment prevent CSC initiation and PanIN formation by regulating glutaminase over-expression, these studies will form a foundation for use of metformin for potential chemoprevention of pancreatic cancer in high risk individuals.